Optical diagnostic assays are utilized to qualitatively and quantitatively detect chemical, biochemical, biological, cellular, and/or particulate species, sometimes referred to herein as analytes, in biological, clinical, environmental, or foodstuff samples. Performing such assays has been especially helpful, for example, in monitoring growing cell cultures and identifying and counting the number of particular cells of interest, such as cancer cells in a blood sample. In general, an assay consists of infusing selected fluorescent dyes, sometimes referred to herein as fluorophores, in a sample of a culture of interest to label and reveal cell types, from which to diagnose, investigate or monitor the progress of a specified disease and the extent to which a particular treatment regimen is successful (or ineffective) to arrest the disease in a human or animal subject.
The '300 provisional application addresses a need for simple, portable, inexpensive, and integrated assay and diagnostic approaches that are appropriate for use in minimal infrastructure, resource-limited settings such as those found in the developing world. Similarly, this need exists for use in resource-limited environments such as those encountered by emergency first responders, primary care physicians, patients at home, forensic investigators, and military field personnel. Optical detection approaches based on fluorescence, absorbance, or luminescence are frequently used methods in well-funded clinical and industrial laboratory analyses to quantify particulate, chemical or biochemical analytes including cells, subcellular components, and biomolecules. But such methods have not found widespread application in those resource-limited settings and environments primarily because of the complexity and cost of readout hardware for optical assays. In providing the improved assay and diagnostic approaches, the invention disclosed in the '300 provisional application offers advantages of considerably less complex and lower cost in medical and veterinary diagnostics, food safety and environmental testing, and other generalized analyte assessment at wide-ranging points-of-use, without suffering loss of accuracy.
Detection and readout of fluorescence-based assays utilizing typical stokes shift fluorescent reporter elements (fluorophores, or fluorescent chromophores) require detecting signals of relatively long wavelength (red-shifted) emission radiation from excited fluorophores. A problem in achieving the desired detection and readout arises from a need to do so amid a substantial background of comparatively shorter wavelength (blue-shifted) radiation used to excite the fluorphores. The excitation radiation is directed along or scattered towards the axis of detection of samples being assayed. And that radiation may be brighter than the fluorescence emitted from labeled analytes. To prevent the excitation radiation from overwhelming the emitted fluorescence and thereby to facilitate fluorescence detection, it is common practice to employ multiple light directing and filtering elements in the optical path between the assay sample and the detector. These elements are typically found in commercially available fluorescent microscopes, flow cytometers, microplate readers, and other fluorescent assay readout and detection systems.
With reference to FIG. 1, this array of prior art optical hardware 10 generally comprises the following basic optical components. A first bandpass filter (excitation filter) 11 allows passage of radiation wavelengths 15 from a broadband (white light) source 12, for exciting fluorophores in a sample 13 being assayed. A second bandpass filter (emission or barrier filter) 14 limits passage of radiation wavelengths 16 from the sample 13 corresponding to the fluorescent emissions 18 of the fluorphores employed to label the sample. A dichroic mirror 17 assists spatial separation of the excitation and emission radiation paths, the former path extending through an objective lens 19, and the latter path extending through an ocular lens 8 to a detector 9. These optical components are typically fabricated using one to five millimeter-thick glass substrates and contained within a filter cube, block, or wheel with characteristic dimensions of between two and ten centimeters. Typically, they are relatively fragile, bulky and expensive.
Fluorophores commonly used in readout and detection systems of the prior art optical hardware are excited primarily in the range between 359 nanometers (nm) (DAPI) and 649 nm (Cy5). The readout systems excite such fluorescent reporter elements (fluorophores) using broadband sources such as halogen lamps and short arc mercury or xenon gas discharge lamps, which are relatively inefficient from the standpoints of both bandwidth emission and power consumption. The wavelengths produced by these broadband sources range across a spectrum far wider than is needed to excite typical fluorophores. For example, a commonly used xenon arc lamp emits wavelengths greater than 700 nanometers, whereas nominal 30 nm-wide wavelength bands would serve for fluorescence excitation. By way of illustration, a portion of the graph of FIG. 7 (to be discussed in more detail presently) denotes the input power and total radiation of a mercury (hg) short arc source utilized for excitation of fluorescent reporter elements in some prior art systems. Although this source consumes 100 watts (W) of input power, it produces only about 0.1 W total radiation (i.e., 0.1% luminous efficiency, which constitutes the ratio of total luminous flux emitted to total input power) in the 460-500 nm blue band (a nominal 40 nm bandwidth) for FITC excitation. And filament, plasma, or gas broadband sources emit at typical overall luminous efficiencies in a somewhat better, but still power-wasteful range of from about 3.5% to about 8%.
Typical broadband sources employed in prior art readout and detection systems for optical assay of human and animal cells and other analytes are also characterized by relatively large size and considerable heat generation. These characteristics necessitate source placement in a housing sufficiently removed from the sample being analyzed to prevent deleterious effect on the resulting analysis. Similarly, the source must be suitably spaced from the collector, other lenses, fiber-optics, or other means utilized to transmit excitation radiation to the sample. The effect of this relatively wide spacing between elements is transmission loss and index mismatch that further exacerbate luminous efficiency of the overall system.
In general, medicine has traditionally employed application-specific hardware and software, often to perform computer-based analyses for diagnostic procedures. More recently, existing consumer platforms are being considered for medical applications. For example, consumer products such as mobile phones, smartphones, compact digital cameras, tablet computers and laptop computers (in particular, iPHONE® smartphones, WINDOWS® smartphones, ANDROID®-based smartphones and BLACKBERRY® smartphones, iPAD® and other tablet computers, PC (personal computer) and MAC® laptop computers—the marks designated by the superscript symbol ® are registered trademarks of their respective owners for the respective products generically listed immediately following the respective appearances of the marks) within the category of mobile electronic devices continue to progress in embedded computing power and memory. The increasingly rapid pace at which these improvements occur has been dramatic, and has not escaped the attention of practitioners concerning the potential use of such platforms in medical applications. Device designers and manufacturers have exponentially increased the digital imaging systems within internet accessible mobile phones and other consumer electronic devices. Currently, image sensors (also known as photosensor arrays, or camera chips) with resolution of more than 20-megapixels are available to consumers on a widespread basis.
It is a principal aim of the present invention to provide a compact, portable, handheld optical assay system by combining or coupling an optical assay apparatus with a mobile electronic device in a synergistic manner to provide improved capabilities for performing optical assays. Mobile electronic devices are often capable of wirelessly accessing the internet or other communication networks, as well as the cloud. Thus, an optical assay system resulting from the coupling of an optical assay apparatus to such a wireless mobile electronic device would enable assay systems capable of performing assays at discrete points of need, and yet also able to exchange information via global communication infrastructure.
Moreover, it is a primary goal of the invention to enable the coupled optical assay apparatus to exploit the applications (colloquially referred to as “apps”), functions including image sensing, and battery power of the mobile electronic device, and to enable the coupled device to exploit the results of the assay performed by the optical assay apparatus for analysis, processing, comparison with a standard, viewing, storage and selective transmission thereof to a separate site, whether local or remote.
Another important objective is to enable performance of optical assays with apparatus attachably and removably coupled to widely available conventional mobile electronic devices, for synergy therebetween as well as compactness and portability of the enhanced apparatus, with an additional advantage of suitability for low-cost transport to and use in environments where apparatuses for performing optical diagnostic assays are otherwise unavailable.
Yet another aim of the invention is to provide methods of performing such assays, and methods of coupling the assay components and the mobile electronic device together to attain such capabilities.